PROJECT SUMMARY Posterior capsule opacification (PCO) is the most common and costly vision-disrupting complication of cataract surgery. A stated major research priority of the Lens and Cataract Program at NEI is to study the mechanism of TGF??mediated lens fibrosis in order to develop effective means of preventing PCO. During the past 25 years, we have perfected a serum-free primary chick lens cell culture system (DCDMLs) that has been validated as an appropriate model for the mammalian lens. Using this system, we found that TGF? can induce not only lens cell fibrosis (i.e., epithelial-mesenchymal transition to myofibroblasts; EMyT), but also lens fiber cell differentiation. The latter is a major cause of clinically deleterious PCO. In this application, we propose three novel strategies to prevent PCO that target different pathways. Each has the potential to block the development of fibrotic and/or lens fiber-type PCO without increasing the time, complexity, or cost of current standard cataract surgery. They also provide new clinical applications for approved or investigational human therapeutics, a goal of another NIH program (the drug repurposing/rescue initiative at NCATS). (1) We have made the unprecedented discovery that a small molecule multikinase inhibitor FDA-approved in 2012 to fight leukemia blocks TGF?-induced EMyT and lens fiber cell differentiation in DCDMLs, as well as two other processes essential for the development of PCO (lens cell proliferation and migration). Effective levels of this drug can be loaded into, and be released within an hour from, a standard human intraocular lens (IOL), and were non-toxic in rabbits after either intracameral or intravitreal injection. Aim #1 is to assess the ability of such drug-releasing IOLs to prevent PCO in the most commonly used and accepted preclinical animal model for PCO, namely rabbits subjected to mock cataract surgery. These studies will be conduced in collaboration with Dr. Liliana Werner, a worldwide authority on PCO and its preclinical assessment in rabbits. (2) We have recently discovered that 10/10 small molecule inhibitors of ErbB (EGF) family receptors block TGF? from inducing EMyT in DCDMLs. To our knowledge, these studies are the first to reveal an obligatory cooperation between the TGF? and ErbB pathways in fibrosis in lens cells. Aim #2 is to identify the ErbB receptors and ligands required for this process, the essential first step in elucidating the molecular mechanisms of this interaction. We will also test if an FDA-approved ErbB R inhibitor can be delivered via IOL. (3) An obvious but underappreciated consequence of cataract surgery is that the surviving anterior lens epithelial cells loose almost all of their cell-cell contacts. In non-lens systems, two types of druggable transcriptional effectors have been shown to regulate TGF?-induced fibrosis in a cell density-dependent manner. On the basis of preliminary evidence presented in this application, we propose to carry out the first molecular investigation of the role of MRTF-A (Aim #3A) and YAP/TAZ (Aim #3B) in low density-induced, TGF?-dependent fibrosis in lens cells.